Using the mammalian carbonic anhydrase isozymes as models, we propose to study those selective factors which have shaped the evolution of enzymes during the ascent of the human species, and those mechanisms that are involved in the control of enzyme expression at the cellular level. Our studies will focus on (1) the evolutionary origins of the human carbonic anhydrases; (2) the effect of mutations and evolutionary changes on their regulation and activity; and (3) the control of carbonic anhydrase gene expression during cellular differentiation. Through this variety of studies on the factors influencing the function of enzymes, we hope to gain insights into how genetic changes alter the function of enzymes and possibly result in inherited disorders. The experimental approaches to the evolutionary and structure-function studies will include amino acid sequencing, comparative activity studies, and determinations of stabilities to heat and proteolysis. Marrow erythroid cell cultures, murine virus-induced erythroleukemic cell cultures, and rat hepatoma cell cultures will be used to study erythroid differentiation and hormonal control. The levels of the carbonic anhydrase will be assayed in single cells by immunofluorescence methods, and a radioimmunoassay will be used to measure the levels of the isozymes in cellular lysates.